Phalloidin is an actin-binding toxin whose chemistry and bioactivity have been studied since the early 1900s (see, for example, Wieland, T., (1986) Peptides of Poisonous Amanita Mushrooms. ed.; Springer-Verlag: New York, page 256). Phalloidin binds with high affinity to filamentous actin (F-actin) and lowers the critical concentration of actin polymerization in solution. It has been used extensively to study actin dynamics in vitro, and fluorescent analogs of phalloidin provide highly specific reagents for microscopic visualization of the actin cytoskeleton (see, for example, Pringle, J. R., et al. (1991) Methods Enzymol. 194: 729-731. The natural source of phalloidin, Amanita phalloides, the Death Cap mushroom, lives in a complex ecological relationship with host trees and is widely considered to be uncultivable (Wieland (1986) supra). Pure phalloidin sells for ˜$150 per milligram and its fluorescent conjugates are much more expensive. In our efforts to develop high-throughput cell-based screens for compounds that modulate actin cytoskeletal morphology, we have sought an inexpensive source of fluorescently labeled phalloidin. Although there have been a number of syntheses of phalloidin analogs both in solution and on the solid phase, no synthetic route has been published with yields significant enough to provide this reagent in practical quantities. These syntheses reported yields ranging from 0.5% to 1.3% and relied on the preparation of relatively complex building blocks in solution. (See Wulf, E. et al. (1979) Proc. Natl. Acad. Sci., 76: 4498-4502; Falcigno, L. et al. (2001) Chemistry—A European Journal, 7: 4665-4673; Zanotti, G. et al. (2001) Chem. Eur. J. 7: 1479-1485; and Anderson, M. O. and Guy, R. K., (2005) J. Org. Chem. 70: 4578-4584.)
Phalloidin is a bicyclic heptapeptide that contains an unusual bridging thioether linkage between the Cys and Trp residues. The natural product contains four common L-amino acids, a D-threonine residue, an unusual γ,δ-dihydroxy-L-leucine residue, and the rare cis epimer of 3-hydroxy-L-proline. Structure-activity studies have shown that the γ,δ-dihydroxy-L-leucine side chain is not essential for actin binding (see Anderson, M. O. and Guy, R. K., (2005) supra; and Wieland, T., (1983) Int. J. Pept. Protein Res., 22: 257-276).
In efforts to develop high-throughput cell-based screens for compounds that modulate actin cytoskeletal morphology, an inexpensive source of fluorescently labeled phalloidin has been sought.
It is desirable to provide improved approaches, including both compounds and methods for their synthesis, for use in the study of the cytoskeleton and cellular morphology and for developing compounds for treating patients suffering from liver failure due to consumption or ingestion of phalloidin and related compounds.